A hemoglobin-based oxygen carrier for clinical use remains elusive, primarily because cell-free hemoglobin induces vasoconstriction and reduced tissue oxygenation. We propose that vasoconstriction produced by cell-free hemoglobin is the result of normal autoregulatory mechanisms which are engaged in response to increased diffusive O2 transport when hemoglobin is free in the plasma space. We have discovered that the critical properties of cell-free hemoglobin responsible for this diffusive phenomenon, (ligand affinity, viscosity and colloid osmotic pressure) markedly influence the ability of cell-free hemoglobin to deliver O2 to tissue. Different types of chemical modification of hemoglobin elicit a range of physiological effects from vasoactivity and acid-base status to changes in blood volume in animals. These findings have led us to question the long-standing assumption that for cell-free hemoglobins to effectively transport O2, they must mimic the properties of RBCs. We will evaluate key physical and biochemical characteristics of modified hemoglobin solutions to identify pertinent properties that determine physiologic differences in O2 transport. Specific focus will be on ligand binding (O2 and NO), solution properties (viscosity and oncotic pressure), and molecular size. The proposal contains 4 Specific Aims. 1) To select and characterize model cell-free hemoglobins for study on the basis of ligand binding properties believed to be critical determinants of O2 transfer to tissue. These include O2 binding and NO reactivity at the hemes and as nitrosothiol. 2) To select and characterize model cell-free hemoglobins for study on the basis of solution properties believed to be critical determinants of O2 transfer to tissue. These include viscosity, colloid oncotic pressure, molecular weight and molar volume, and the influence of the type of modification (intramolecular versus surface). 3) To use these model cell-free oxygen carriers in an artificial capillary system to determine the alteration to 02 diffusion resulting from the physicochemical properties of the carriers. This system is independent of biological regulatory mechanisms. 4) To measure in vivo hemodynamic cardiac output, acid-base balance, lactate production, and blood volume responses to isovolemic exchange transfusion with the model carriers and subsequent hemorrhage in rats. The effect of the selected carriers on mean arterial blood pressure and cardiac output will provide indications of vasoactive response to hemodilution with these cell-free oxygen carriers in intact animals. Blood volume will be monitored to provide an indication of the fluid shifts that occur in response to isovolemic exchange transfusion. Arterial lactic acid and acid-base status provides additional indicators of the balance between O2 supply and demand. Taken together, these aims will not only lead to rationally-designed oxygen carriers for clinical use, but also important insight into mechanisms of vasoactivity and control of tissue oxygenation.